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C F Meares

Publications and source records attributed to C F Meares.

At least 19 recordsLinked to original sources
PubMed2001

Restructuring of an RNA polymerase holoenzyme elongation complex by lambdoid phage Q proteins.

The structure of an intermediate in the initiation to elongation transition of Escherichia coli RNA polymerase has been visualized through region-specific DNA cleavage by the hydroxyl radical reagent FeBABE. FeBABE was tethered to specific sites of the final sigma(70) subunit and incorporated into two specialized paused elongation complexes that obligatorily retain the final sigma(70) initiation subunit and are targets for modification by lambdoid phage late gene antiterminators. The FeBABE cleavage pattern reveals structures similar to open complex, except for notable changes to region 3 of final sigma(70) that might reflect the presence of stably bound transcript. Binding of the antiterminator protein Q displaces the reactivity of FeBABE conjugated to region 4 of final sigma(70), suggesting that final sigma(70) subunit rearrangement is a step in conversion of RNAP to the antiterminating form.

Bacteriophage lambda↗
PubMed2001

Antibodies with infinite affinity.

Here we report an approach to the design and production of antibody/ligand pairs, to achieve functional affinity far greater than avidin/biotin. Using fundamental chemical principles, we have developed antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. Choosing a structurally characterized antibody/ligand pair as an example, we engineered complementary reactive groups in the antibody binding pocket and the ligand, so that they would be in close proximity in the antibody/ligand complex. Cross-reactions with other molecules in the medium are averted because of the low reactivity of these groups; however, in the antibody/ligand complex the effective local concentrations of the complementary reactive groups are very large, allowing a covalent reaction to link the two together. By eliminating the dissociation of the ligand from the antibody, we have made the affinity functionally infinite. This chemical manipulation of affinity is applicable to other biological binding pairs.

Animals↗
PubMed2001

Helix packing in the lactose permease of Escherichia coli: distances between site-directed nitroxides and a lanthanide.

By exploiting substrate protection of Cys148 in lactose permease, a methanethiosulfonate nitroxide spin-label was directed specifically to one of two Cys residues in a double-Cys mutant, followed by labeling of Cys148 with a thiol-reactive chelator that binds Gd(III) quantitatively. Distances between bound Gd(III) and the nitroxide spin-label were then studied by electron paramagnetic resonance. The results demonstrate that the Gd(III)-induced relaxation effects on nitroxides at positions 228, 226 (helix VII), and 275 (helix VIII) agree qualitatively with results obtained by studying spin-spin interactions [Wu, J., Voss, J., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 10123-10127]. Thus, a nitroxide attached to position 228 (helix VII) is closest to the lanthanide at position 148 (helix V), a nitroxide at position 275 (helix VIII) is further away, and the distance between positions 226 (helix VII) and 148 is too long to measure. However, the Gd(III)-spin-label distances are significantly longer than those estimated from nitroxide-nitroxide interactions between the same pairs due to the nature of the chelator. Although the results provide strong confirmation for the contention that helix V lies close to both helices VII and VIII in the tertiary structure of lactose permease, other methods for binding rare earth metals are discussed which do not involve the use of bulky chelators with long linkers.

Binding Sites↗
PubMed2001

Advances in pretargeting biotechnology.

A major focus of current drug research is to improve drug targeting to internal target sites such as to solid tumors or specific organs. The objective of drug targeting, especially for cancer chemotherapy and radioimmunotherapy, is to enhance the effectiveness of the drug by concentrating it at the target site and minimizing its effects in nontarget sites. Although tumor targeting has been obtained with large long-circulating radiolabeled antibody molecules, normal organ activity, especially in the blood kidneys, liver, and bone marrow is a significant problem. Over the last 20 years, studies to improve the therapeutic use of antibodies have included the use of antibody fragments, chase molecules, metabolizable linkers, antibody-directed enzyme prodrugs (ADEPT), local delivery, and pretargeting. Here, we will review the most interesting recent advances in pretargeting biotechnology.

Journal Article↗
PubMed2001

Biodistribution and dosimetry of pretargeted monoclonal antibody 2D12.5 and Y-Janus-DOTA in BALB/c mice with KHJJ mouse adenocarcinoma.

UNLABELLED: Biodistribution and dosimetry of 88Y (and equimolar 90Y) Janus-dodecanetetraacetic acid (DOTA) were performed using a three-step pretargeting protocol in BALB/c mice bearing mouse mammary adenocarcinoma (KHJJ) implants. Pretargeting was performed with mouse monoclonal antibody (mAb) 2D12.5 specific for yttrium-DOTA, and the chase was Y-DOTA-human transferrin conjugate. In this article, we report extensive organ dosimetry and the theoretic limits of the radionuclide physical half-life (T(p)) for pretargeting. METHODS: Organ biodistribution data were obtained from bioassays on tissue taken from tumor mice killed at 3, 24, 48, 72, 96, and 120 h after intravenous injection of 88Y-Janus-DOTA. Uptake and retention of 88Y as a function of time were described by nonlinear least squares fits of the tissue data to multiexponential functions. Radiation dose estimates for equivalent molar amounts of 90Y were subsequently derived from these time-integrated functions. RESULTS: The results were as follows: rapid blood clearance of 88Y-Janus-DOTA; rapid uptake and slow clearance of 88Y-Janus-DOTA from the tumor over 5 d; rapid clearance from all organs and body; largest radiation absorbed dose (AD) per injected dose of 63.52 (cGy/MBq) to tumor; high therapeutic ratios (AD tumor/AD tissue), particularly for blood and bone; and optimal radionuclide T(p) range from 30 min to 10 d. CONCLUSION: Although the absolute concentration of 90Y in the tumor is less using the hapten system than is achieved generally with the chelated radionuclide covalently attached to the mAb, the achievable tumor uptake of radioactivity, coupled with low radioactivity in bone, blood, and other organs, suggests that a three-step pretargeting protocol has considerable promise as a method for 90Y radioimmunotherapy.

Adenocarcinoma↗
PubMed2001

Metabolite production in patients with lymphoma after radiometal-labeled antibody administration.

UNLABELLED: Radiometal-labeled monoclonal antibodies are retained longer in tumors than iodinated antibodies, leading to their increased use for radioimmunotherapy. Dissociation of radioiodine from the antibody during metabolism has been documented. We now report metabolites in the plasma of lymphoma patients given 111In- and 90Y-2-iminothiolane-2-[p-(bromoacetamido)benzyl]-1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid-Lym-1 (111In/90Y-2IT-BAD-Lym-1). METHODS: Nineteen patients with non-Hodgkin's lymphoma (NHL) received 111In- and 90Y-2IT-BAD-Lym-1; 111In was used as a surrogate tracer for 90Y, which emits no gamma-photon. Plasma was obtained up to 7 d and analyzed by high-performance liquid chromatography to determine the fraction of radiolabel associated with monomeric antibody, metabolites, and complexed antibody. Planar images of conjugate views were acquired up to 7 d and used to quantitate 111In in organs and tumors. RESULTS: Metabolites and complexes were observed in the plasma of every patient who received 111In-2IT-BAD-Lym-1. At 3 d, the mean percentages of 111In in the patients' plasma in monomeric, metabolite, and complexed forms were 54%, 36%, and 10%, respectively. Metabolites of 90Y-2IT-BAD-Lym-1 were formed to a similar extent. In comparison, in groups of breast and prostate cancer patients who received the radioimmunoconjugate 111In-2IT-BAD-m170, 91% and 94% of 111In in the patients' plasma were in monomeric form, respectively. Metabolites and complexes of 111In-2IT-BAD-Lym-1 contributed a mean 10% of the total area under the time-activity curve (AUC) for blood. Little formation of metabolites and complexes occurred in vitro in NHL patient or volunteer plasma or in Raji cell culture. The clinical and in vitro data supported the processing of 111In/90Y-2IT-BAD-Lym-1 in the hepatocytes as the dominant mechanism for the production of metabolites. CONCLUSION: Metabolites of 111In/90Y-2IT-BAD-Lym-1 accounted for 10% of blood AUC in patients. The therapeutic index was adversely affected by metabolism of 111In/90Y-2IT-BAD-Lym-1 to the extent that the tumor specificity of the radioactive metabolites was lost.

Adult↗
PubMed2000

Protein-protein interactions mapped by artificial proteases: where sigma factors bind to RNA polymerase.

Interactions between proteins are important to understand but difficult to study. Conjugating a protein to a small artificial protease endows it with the ability to cut other proteins where it binds to them. Analysing the sites cut on the target proteins leads to new understanding of the structure of each complex. The binding of sigma factors to a common region on RNA polymerase provides an example.

Bacterial Proteins↗
PubMed2000

Physical and chemical properties of radionuclide therapy.

As more radionuclide therapies move from laboratory feasibility studies into clinical reality, it becomes increasingly important for the labeling chemistry to produce consistently a stable radiopharmaceutical that remains intact under the challenge of human catabolism. Similarly, once proof of principle is established to bring a radionuclide conjugate into clinical therapy trials, dosimetric estimates should be made to select the appropriate radionuclide properties, which are based on animal-specific or patient-specific pharmacokinetics and match a set of specific clinical endpoints. These properties may include the radionuclide physical half-life, radiolabeled conjugate biological uptake and clearance, product-specific activity, range and type of emissions, and resultant effects on tumor and normal tissue cellular survival. The immunologist and labeling chemist have now produced a variety of strategies that have potential to increase the therapeutic ratio (tumor-to-normal tissue dose ratio). The advent of normal tissue clearing agents, fragmented or chimerized carriers to improve targeting, and the method of bispecific or two-step and three-step targeting agents has increased the need for realistic modeling of the carrier in vivo to guide prospectively the competitive development of these radiopharmaceuticals. In this article, examples have been taken from the literature to elucidate the benchmark of success that careful experimental design has fostered to bring these agents into clinical practice by creative and logical methodologies.

Half-Life↗
PubMed2000

Positioning of region 4 of the Escherichia coli RNA polymerase sigma(70) subunit by a transcription activator.

A DNA cleavage reagent, specifically tethered to residue 581 of the Escherichia coli RNA polymerase sigma(70) subunit, has been used to investigate the location of sigma(70) region 4 in different complexes at the galp(1) promoter and the effect of the cyclic AMP receptor protein. The positions of DNA cleavage by the reagent are not affected by the cyclic AMP receptor protein. We conclude that transcription activation at the galp(1) promoter by the cyclic AMP receptor protein does not involve major conformation changes in or repositioning of sigma(70) region 4.

Bacterial Proteins↗
PubMed2000

Are radiometal-labeled antibodies better than iodine-131-labeled antibodies: comparative pharmacokinetics and dosimetry of copper-67-, iodine-131-, and yttrium-90-labeled Lym-1 antibody in patients with non-Hodgkin's lymphoma.

Radioimmunotherapy using radiolabeled monoclonal antibodies against tumor-associated antigens has been efficacious, particularly in the treatment of radiosensitive malignancies such as lymphoma. Antilymphoma monoclonal antibody Lym-1, labeled with copper-67 ((67)Cu), iodine-131 ((131)I), or yttrium-90 ((90)Y), has been effective salvage therapy for patients with non-Hodgkin's lymphoma. Although (131)I has had the dominant role in radioimmunotherapy thus far, several properties of radiometals are preferable. A total of 70 patients with B-lymphocytic non-Hodgkin's lymphoma were studied using (67)Cu-2IT-BAT-Lym-1, (131)I-Lym-1, or (111)In-2IT-BAD-Lym-1. Because (90)Y does not have good emissions for imaging, indium-111 ((111)In), its analogue, was used as a surrogate to estimate (90)Y-2IT-BAD-Lym-1 pharmacokinetics and radiation dosimetry. Subsets of four patients in each group received (67)Cu- and (131)I-labeled Lym-1 or (111)In- and (131)I-labeled Lym-1, allowing direct comparisons of the radioimmunoconjugates. Sequential blood samples and planar images were used to quantitate radioimmunoconjugate in tissues in order to determine pharmacokinetics and radiation dosimetry. (67)Cu-2IT-BAT-Lym-1 and (90)Y-2IT-BAD-Lym-1 exhibited higher cumulated activity concentrations and radiation absorbed doses per unit of administered radioactivity for tumors than did (131)I-Lym-1. The mean tumor cumulated activity (area under the time-activity curve) concentrations per unit of administered radioactivity for (67)Cu-2IT-BAT-Lym-1, (131)I-Lym-1, and (90)Y-2IT-BAD-Lym-1 were 96.89, 33.96, and 43.42 GBq-s/GBq/g, respectively. The mean tumor radiation doses from (67)Cu-2IT-BAT-Lym-1, (131)I-Lym-1, and (90)Y-2IT-BAD-Lym-1 were 2.5, 1.0, and 6.6 Gy/GBq, respectively, because (90)Y deposits more radiation per unit of administered radioactivity. Per unit of administered radioactivity, radiation doses from (67)Cu-2IT-BAT-Lym-1 and (131)I-Lym-1 to normal tissues were similar except that the liver received a higher dose from (67)Cu-2IT-BAT-Lym-1 than from (131)I-Lym-1; radiation doses to normal tissues from (90)Y-2IT-BAD-Lym-1 were generally higher. Consequently, the therapeutic indices (ratio of radiation doses to tumor and normal tissues) for (67)Cu-2IT-BAT-Lym-1, and less generally for (90)Y-2IT-BAD-Lym-1, were more favorable when compared to those for (131)I-Lym-1. Data from the matched subsets of patients showed similar therapeutic indices to those for the groups of patients. (67)Cu-2IT-BAT-Lym-1 showed more potential than (131)I-Lym-1 or (90)Y-2IT-BAD-Lym-1 for non-Hodgkin's lymphoma radioimmunotherapy.

Adult↗
PubMed2000

Radiation dosimetry for 90Y-2IT-BAD-Lym-1 extrapolated from pharmacokinetics using 111In-2IT-BAD-Lym-1 in patients with non-Hodgkin's lymphoma.

UNLABELLED: Several monoclonal antibodies, including Lym-1, have proven effective for treatment of hematologic malignancies. Lym-1, which preferentially targets malignant lymphocytes, has induced therapeutic responses and prolonged survival in patients with non-Hodgkin's lymphoma (NHL) when labeled with 131. Because radiometal-labeled monoclonal antibodies provide higher tumor radiation doses than corresponding 131I-labeled monoclonal antibodies, the radiation dosimetry of 90Y-2-iminothiolane-2-[p-(bromoacetamido)benzyl]-1,4,7,10-tetraazacyc lododecane-N,N',N",N"'-tetraacetic acid-Lym-1 (90Y-21T-BAD-Lym-1) is of importance because of its potential for radioimmunotherapy. Although 90Y has attractive properties for therapy, its secondary bremsstrahlung is less suitable for imaging and pharmacokinetic studies in patients. Thus, the pharmacokinetic data obtained for 111In-21T-BAD-Lym-1 in patients with NHL were used to calculate dosimetry for 90SY-21T-BAD-Lym-1. METHODS: Thirteen patients with advanced-stage NHLwere given a preload dose of unmodified Lym-1 followed by an imaging dose of 111In-21T-BAD-Lym-1. Sequential imaging and blood and urine samples obtained for up to 10 d after infusion were used to assess pharmacokinetics. Using 111In pharmacokinetic data and 90Y physical constants, radiation dosimetry for 90Y-21T-BAD-Lym-1 was determined. RESULTS: The uptake of 111In-21T-BAD-Lym-1 in tumors was greater than uptakes in the lung and kidney but similar to uptakes in the liver and spleen. The biologic half-time in tumors was greater than in lungs. The mean radiation dose to tumors was 6.57 +/- 3.18 Gy/GBq. The mean tumor-to-marrow (from blood) radiation ratio was 66:1, tumor-to-total body was 13:1, and tumor-to-liver was 1:1. Images of 111In were of excellent quality; tumors and normal organs were readily identified. Mild and transient Lym-1 toxicity occurred in 3 patients. CONCLUSION: Because of the long residence time of 111In-2IT-BAD-Lym-1 in tumors, high 90Y therapeutic ratios (tumor-to-tissue radiation dose) were achieved for some tissues, but the liver also showed high uptake and retention of the radiometal.

Adult↗
PubMed1999

Targeted protein footprinting: where different transcription factors bind to RNA polymerase.

Gene transcription is regulated through the interactions of RNA polymerase (RNAP) with transcription factors, such as the bacterial sigma proteins. We have devised a new strategy that relies on targeted protein footprinting to make an extensive survey of proximity to the protein surface. This involves attaching cutting reagents randomly to lysine residues on the surface of a protein such as sigma. The lysine-labeled sigma protein is then used to cleave the polypeptide backbones of the RNAP proteins at exposed residues adjacent to the sigma binding site. We used targeted protein footprinting to compare the areas near which sigma(70), sigma(54), sigma(38), sigma(E), NusA, GreA, and omega bind to the protein subunits of Escherichia coli RNAP. The sigma proteins and NusA cut sites in similar regions of the two large RNAP subunits, beta and beta', outlining a common surface. GreA cuts a larger set of sites, whereas omega shows no overlap with the others, cutting only the beta' subunit at a unique location.

Bacterial Proteins↗
PubMed1999

Positioning of sigma(S), the stationary phase sigma factor, in Escherichia coli RNA polymerase-promoter open complexes.

The sigma(S) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and is required for promoter recognition of many stationary phase genes. We have analysed open complexes of Esigma(S) RNA polymerase, using sigma(S) derivatives carrying single cysteine residues at nine different positions to which the reagent FeBABE has been tethered. All holoenzymes but one formed transcriptionally active open complexes at three different promoters (osmY, galP1 and lacUV5). The chemical nuclease FeBABE can cleave DNA in proximity to the chelate. The overall cutting pattern of Esigma(S) open complexes does not depend on the nature of the promoter and is similar to that obtained with Esigma(70), but extends towards the downstream part of the promoter. The strongest cleavages are observed with FeBABE positioned on cysteines in regions 2.2 to 3.1. In contrast to sigma(70), region 2.1 of sigma(S) appears to be far from DNA. Region 4.2 of sigma(S) appears less accessible than its counterpart in sigma(70) and FeBABE positioned in the turn of the helix-turn-helix (HTH) motif in region 4.2 reacts only weakly with the -35 promoter element. This provides a structural basis for the minor role of the -35 sequence in sigma(S)-dependent promoter recognition.

Amino Acid Substitution↗
PubMed1999

Mapping protein-protein interactions with a library of tethered cutting reagents: the binding site of sigma 70 on Escherichia coli RNA polymerase.

Surface-exposed lysine amino groups and other reactive nucleophiles of the sigma 70 protein were conjugated with the cutting reagent iron (S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (FeBABE) via 2-iminothiolane (2IT) with low efficiency. The result is a library of sigma 70 conjugates, with an average of 1-2 cutting reagents tethered to any of a variety of sites (lysine, cysteine, etc.) on the surface of the protein. Model calculations indicate that the conjugates in this library should be capable of cutting nearby sites on the backbone of almost any protein or nucleic acid to which sigma 70 binds. Since cutting occurs only when the protein is bound, the cleaved sites indicate proximity; since only proximal sites are cleaved, interpretation of the results is straightforward. We used this library to map the periphery of the binding site on the core enzyme (alpha 2 beta beta') of Escherichia coli RNA polymerase. The beta subunit was cut primarily within its conserved regions C, D, Rif I, and G; additional sites were also cut between A and B and near conserved regions E and H. The cut sites within the beta' subunit were intensely clustered between residues 250-450, which include its conserved regions C and D, along with two additional cut sites in conserved regions A and G. No cut sites on the alpha subunit were observed. These results recapitulate and extend those obtained using FeBABE conjugates of seven strategically placed single-Cys sigma 70 mutants [Owens, J. T., Miyake, R., Murakami, K., Chmura, A. J., Fujita, N., Ishihama, A., and Meares, C. F. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6021-6026]. This technique provides a straightforward, general approach to mapping protein interactions without mutagenesis.

Binding Sites↗
PubMed1999

Organization of open complexes at Escherichia coli promoters. Location of promoter DNA sites close to region 2.5 of the sigma70 subunit of RNA polymerase.

A cysteine-tethered DNA cleavage agent has been used to locate the position of region 2.5 of sigma70 in transcriptionally competent complexes between Escherichia coli RNA polymerase and promoters. In this study we have engineered sigma70 to introduce a unique cysteine residue at a number of positions in region 2.5. Mutant proteins were purified, and in each case, the single cysteine residue used as the target for covalent coupling of the DNA cleavage agent p-bromoacetamidobenzyl-EDTA.Fe (FeBABE). RNA polymerase core reconstituted with tagged sigma derivatives was shown to be transcriptionally active. Hydroxyl radical-based DNA cleavage mediated by tethered FeBABE was observed for each derivative of RNA polymerase in the open complex. Our results show that region 2.5 is in close proximity to promoter DNA just upstream of the -10 hexamer. This positioning is independent of promoter sequence. A model for the interaction of this region of sigma with promoter DNA is discussed.

Base Sequence↗
PubMed1999

Transfer of copper from a chelated 67Cu-antibody conjugate to ceruloplasmin in lymphoma patients.

The Lym-1 monoclonal antibody was conjugated with the bifunctional chelating agent 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N ',N'',N'''-tetraacetic acid (BAT), using 2IT as a linker, and radiolabeled with 67Cu to make the radiopharmaceutical, 67Cu-2IT-BAT-Lym-1. Ten patients received a total of 18 doses of 67Cu-2IT-BAT-Lym-1 as targeted, systemic radiotherapy. The beta phase of blood clearance, when corrected for 67Cu decay, was positive or flat, a phenomenon not observed in similar patients treated with 131I-Lym-1. The flat beta phase of blood clearance suggested recycling of 67Cu from 67Cu-2IT-BAT-Lym-1 to another plasma protein. Therefore, the amount of 67Cu transferred from the radiopharmaceutical to CP, Alb, and TF was measured using affinity-purified polyclonal antibodies. The fraction of plasma 67Cu precipitated by anti-human CP increased daily; most blood radioactivity was 67Cu-CP after a median of 4 days (range 2-7 days). The transfer of 67Cu to CP was observed in all patients and was consistent from dose to dose within the same patient. An average of 2.8 +/- 1.5% (range 0.8-7.8%) of the 67Cu dose (%ID) was transferred to CP. The release rate of 67Cu-CP from the liver into the blood was 0.9 +/- 0.4 %ID/day for the first 3 days. The 67Cu-CP effective clearance half-life was 3.7 +/- 0.7 days. Subtraction of the 67Cu-CP activity from the total blood radioactivity yielded a biphasic blood clearance similar to that obtained for patients given 131I-Lym-1. Cu-67-CP increased the AUC for whole blood by 24 +/- 10%. The %ID of 67Cu recycled correlated with GGT, ALT, and alkaline phosphatase levels; r = 0.958 (p < 0.001), 0.857 (p < 0.01), and 0.822 (p < 0.01), respectively. Albumin levels correlated negatively with recycled copper (r = -0.745, p < 0.05). The data suggest that the liver metabolizes 67Cu-2IT-BAT-Lym-1 and recycles a small fraction of the 67Cu, transferring it to CP.

Adult↗